Human G-protein receptor HGBER32

ABSTRACT

Human G-protein coupled receptor polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed were methods for utilizing such polypeptides for identifying antagonists and agonists to such polypeptides and methods of using the agonists and antagonists therapeutically to treat conditions related to the underexpression and overexpression of the G-protein coupled receptor polypeptides, respectively. Also disclosed are diagnostic methods for detecting a mutation in the G-protein coupled receptor nucleic acid sequences and an altered level of the soluble form of the receptors.

[0001] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptide ofthe present invention is a human 7-transmembrane receptor. Thetransmembrane receptor is a G-protein coupled receptor. Moreparticularly, the 7-transmembrane receptor has been putativelyidentified as a G-protein chemokine receptor, sometimes hereinafterreferred to as “HGBER32”. The invention also relates to inhibiting theaction of such polypeptides.

[0002] It is well established that many medically significant biologicalprocesses are mediated by proteins participating in signal transductionpathways that involve G-proteins and/or second messengers, e.g., cAMP(Lefkowitz, Nature, 351:353-354 (1991)). Herein these proteins arereferred to as proteins participating in pathways with G-proteins or PPGproteins. Some examples of these proteins include the GPC receptors,such as those for adrenergic agents and dopamine (Kobilka, B. K., etal., PNAS, 84:46-50 (1987); Kobilka, B. K., et al., Science, 238:650-656(1987); Bunzow, J. R., et al., Nature, 336:783-787 (1988)), G-proteinsthemselves, effector proteins, e.g., phospholipase C, adenyl cyclase,and phosphodiesterase, and actuator proteins, e.g., protein kinase A andprotein kinase C (Simon, M. I., et al., Science, 252:802-8 (1991)).

[0003] For example, in one form of signal transduction, the effect ofhormone binding is activation of an enzyme, adenylate cyclase, insidethe cell. Enzyme activation by hormones is dependent on the presence ofthe nucleotide GTP, and GTP also influences hormone binding. A G-proteinconnects the hormone receptors to adenylate cyclase. G-protein was shownto exchange GTP for bound GDP when activated by hormone receptors. TheGTP-carrying form then binds to an activated adenylate cyclase.Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns theG-protein to its basal, inactive form. Thus, the G-protein serves a dualrole, as an intermediate that relays the signal from receptor toeffector, and as a clock that controls the duration of the signal.

[0004] The membrane protein gene superfamily of G-protein coupledreceptors has been characterized as having seven putative transmembranedomains. The domains are believed to represent transmembrane α-helicesconnected by extracellular or cytoplasmic loops. G-protein coupledreceptors include a wide range of biologically active receptors, such ashormone, viral, growth factor and neuroreceptors.

[0005] G-protein coupled receptors have been characterized as includingthese seven conserved hydrophobic stretches of about 20 to 30 aminoacids, connecting at least eight divergent hydrophilic loops. TheG-protein family of coupled receptors includes dopamine receptors whichbind to neuroleptic drugs used for treating psychotic and neurologicaldisorders. Other examples of members of this family include calcitonin,adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine,serotonin, histamine, thrombin, kinin, follicle stimulating hormone,opsins, endothelial differentiation gene−1 receptor and rhodopsins,odorant, cytomegalovirus receptors, etc.

[0006] Most G-protein coupled receptors have single conserved cysteineresidues in each of the first two extracellular loops which formdisulfide bonds that are believed to stabilize functional proteinstructure. The 7 transmembrane regions are designated as TM1, TM2, TM3,TM4, TM5, TM6, and TM7. TM3 (the intracellular loop between TM3 and TM4)has been implicated in signal transduction.

[0007] Phosphorylation and lipidation (palmitylation or farnesylation)of cysteine residues can influence signal transduction of some G-proteincoupled receptors. Most G-protein coupled receptors contain potentialphosphorylation sites within the third cytoplasmic loop and/or thecarboxy terminus. For several G-protein coupled receptors, such as theβ-adrenoreceptor, phosphorylation by protein kinase A and/or specificreceptor kinases mediates receptor desensitization.

[0008] The ligand binding sites of G-protein coupled receptors arebelieved to comprise a hydrophilic socket formed by several G-proteincoupled receptors transmembrane domains, which socket is surrounded byhydrophobic residues of the G-protein coupled receptors. The hydrophilicside of each G-protein coupled receptor transmembrane helix ispostulated to face inward and form the polar ligand binding site. TM3has been implicated in several G-protein coupled receptors as having aligand binding site, such as including the TM3 aspartate residue.Additionally, TM5 serines, a TM6 asparagine and TM6 or TM7phenylalanines or tyrosines are also implicated in ligand binding.Furthermore, the entracellular hydrophilic domains have also been shownto have a role in ligand binding.

[0009] G-protein coupled receptors can be intracellularly coupled byheterotrimeric G-proteins to various intracellular enzymes, ion channelsand transporters (see, Johnson et al., Endoc., Rev., 10:317-331 (1989)).Different G-protein α-subunits preferentially stimulate particulareffectors to modulate various biological functions in a cell.Phosphorylation of cytoplasmic residues of G-protein coupled receptorshave been identified as an important mechanism for the regulation ofG-protein coupling of some G-protein coupled receptors. G-proteincoupled receptors are found in numerous sites within a mammalian host.

[0010] In accordance with one aspect of the present invention, there areprovided novel polypeptides as well as biologically active anddiagnostically or therapeutically useful fragments and derivativesthereof. The polypeptides of the present invention are of human origin.

[0011] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding the polypeptide ofthe present invention including mRNAs, DNAs, cDNAs, genomic DNA as wellas antisense analogs thereof and biologically active and diagnosticallyor therapeutically useful fragments thereof.

[0012] In accordance with a further aspect of the present invention,there is provided a process for producing such polypeptides byrecombinant techniques which comprises culturing recombinant prokaryoticand/or eukaryotic host cells, containing a nucleic acid sequenceencoding a polypeptide of the present invention, under conditionspromoting expression of said polypeptide and subsequent recovery of saidpolypeptide.

[0013] In accordance with yet a further aspect of the present invention,there are provided antibodies against such polypeptides.

[0014] In accordance with another aspect of the present invention thereare provided methods of screening for compounds which bind to andactivate or inhibit activation of the receptor polypeptides of thepresent invention and for receptor ligands.

[0015] In accordance with still another embodiment of the presentinvention there is provided a process of using such activating compoundsto stimulate the receptor polypeptide of the present invention for thetreatment of conditions related to the under-expression of the G-proteincoupled receptors.

[0016] In accordance with another aspect of the present invention thereis provided a process of using such inhibiting compounds for treatingconditions associated with over-expression of the G-protein coupledreceptors.

[0017] In accordance with yet another aspect of the present inventionthere is provided non-naturally occurring synthetic, isolated and/orrecombinant G-protein coupled receptor polypeptides which are fragments,consensus fragments and/or sequences having conservative amino acidsubstitutions, of at least one transmembrane domain of the G-proteincoupled receptor of the present invention, such that the receptor maybind G-protein coupled receptor ligands, or which may also modulate,quantitatively or qualitatively, G-protein coupled receptor ligandbinding.

[0018] In accordance with still another aspect of the present inventionthere are provided synthetic or recombinant G-protein coupled receptorpolypeptides, conservative substitution and derivatives thereof,antibodies, anti-idiotype antibodies, compositions and methods that canbe useful as potential modulators of G-protein coupled receptorfunction, by binding to ligands or modulating ligand binding, due totheir expected biological properties, which may be used in diagnostic,therapeutic and/or research applications.

[0019] It is still another object of the present invention to providesynthetic, isolated or recombinant polypeptides which are designed toinhibit or mimic various G-protein coupled receptors or fragmentsthereof, as receptor types and subtypes.

[0020] In accordance with yet a further aspect of the present invention,there is also provided diagnostic probes comprising nucleic acidmolecules of sufficient length to specifically hybridize to the nucleicacid sequences of the present invention.

[0021] In accordance with yet another object of the present invention,there is provided a diagnostic assay for detecting a disease orsusceptibility to a disease related to a mutation in a nucleic acidsequence of the present invention.

[0022] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

[0023] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0024] FIGS. 1A-1D show the cDNA sequence (SEQ ID NO:1) and thecorresponding deduced amino acid sequence (SEQ ID NO:2) of the HGBER32G-protein coupled receptor of the present invention. The standardone-letter abbreviation for amino acids is used.

[0025]FIG. 2 is an illustration of the secondary structural features ofthe HGBER32 G-protein coupled receptor. The first 7 illustrations setforth the regions of the amino acid sequence which are alpha helices,beta sheets, turn regions or coiled regions. The boxed areas are theareas which correspond to the region indicated. The second set offigures illustrate areas of the amino acid sequence which are exposed tointracellular, cytoplasmic or are membrane-spanning. The hydrophilicityplot illustrates areas of the protein sequence which are the lipidbilayer of the membrane and are, therefore, hydrophobic, and areasoutside the lipid bilayer membrane which are hydrophilic. The antigenicindex corresponds to the hydrophilicity plot, since antigenic areas areareas outside the lipid bilayer membrane and are capable of bindingantigens. The surface probability plot further corresponds to theantigenic index and the hydrophilicity plot. The amphipathic plots showthose regions of the protein sequences which are polar and non-polar.The flexible regions corresond to the second set of illustrations in thesense that flexible regions are those which are outside the membrane andinflexible regions are transmembrane regions.

[0026] FIGS. 3A-3C illustrate an amino acid alignment of the G-proteincoupled receptor of the present invention (HGBER32) beginning at aminoacid 6 and human monocyte chemoattractant protein 1 receptor (MCP-16b)beginning at amino acid 40 (SEQ ID NO:3). Line matches indicateidentical amino acids (40.401%) and dot matches indicate similar aminoacids (64.470%).

[0027]FIG. 4 illustrated the predicted transmembrane domains forHGBER32.

[0028] In accordance with an aspect of the present invention, there areprovided isolated nucleic acids (polynucleotides) which encode for themature polypeptide having the deduced amino acid sequence of FIGS. 1A-1D(SEQ ID NO:2) or for the mature polypeptide encoded by the cDNA of theclone deposited as ATCC Deposit No. 97187 on Jun. 1, 1995

[0029] The polynucleotide of this invention was discovered in a cDNAlibrary derived from human gall bladder tissue. It is structurallyrelated to the G protein-coupled receptor family. It contains an openreading frame encoding a mature protein of 355 amino acid residues. Theprotein exhibits the highest degree of homology to human-monocytechemoattractant protein 1 receptor (MCP-16b) with about 40% identity andabout 64% similarity.

[0030] The polynucleotides of the present invention may be in the formof RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in FIGS. 1A-1D (SEQ ID NO:1) orthat of the deposited clone or may be a different coding sequence whichcoding sequence, as a result of the redundancy or degeneracy of thegenetic code, encodes the same mature polypeptide as the DNA of FIGS.1A-1D (SEQ ID NO:1) or the deposited cDNA.

[0031] The polynucleotides which encode for the mature polypeptides ofFIGS.1A-1D (SEQ ID NO:2) or for the mature polypeptide encoded by thedeposited cDNA may include: only the coding sequence for the maturepolypeptide; the coding sequence for the mature polypeptide (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence 5′ and/or 3′ of the coding sequence forthe mature polypeptide.

[0032] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0033] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptide having the deduced amino acidsequence of FIGS. 1A-1D (SEQ ID NO:2) or the polypeptide encoded by thecDNA of the deposited clone. The variants of the polynucleotides may bea naturally occurring allelic variant of the polynucleotides or anon-naturally occurring variant of the polynucleotides.

[0034] Thus, the present invention includes polynucleotides encoding thesame mature polypeptide as shown in FIGS. 1A-1D (SEQ ID NO:2) or thesame mature polypeptide encoded by the cDNA of the deposited clone aswell as variants of such polynucleotides which variants encode for afragment, derivative or analog of the polypeptide of FIGS. 1A-1D (SEQ IDNO:2) or the polypeptide encoded by the cDNA of the deposited clone.Such nucleotide variants include deletion variants, substitutionvariants and addition or insertion variants.

[0035] As hereinabove indicated, the polynucleotides may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIGS. 1A-1D (SEQ ID NO:1) or of the coding sequence ofthe deposited clone. As known in the art, an allelic variant is analternate form of a polynucleotide sequence which may have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptides.

[0036] The polynucleotides may also encode for a soluble form of thereceptor polypeptide of the present invention which is the extracellularportion of the polypeptide which has been cleaved from the TM andintracellular domain of the full-length polypeptide of the presentinvention.

[0037] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptides of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

[0038] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least70%, preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIGS. 1A-1D (SEQ ID NO:1)or the deposited cDNA(s), i.e. function as a soluble receptorpolypeptide by retaining the ability to bind the ligands for thereceptor even though the polypeptide does not function as a membranebound receptor polypeptide, for example, by eliciting a second messengerresponse.

[0039] Alternatively, the polynucleotide may have at least 20 bases,preferably at least 30 bases, and more preferably at least 50 baseswhich hybridize to a polynucleotide of the present invention and whichhas an identity thereto, as hereinabove described. Such polynucleotidesmay be employed as probes for the polynucleotide of SEQ ID NO:1, forexample, for recovery of the polynucleotide or as a diagnostic probe oras a PCR primer.

[0040] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0041] The present invention further relates to a G-protein coupledreceptor polypeptide which has the deduced amino acid sequence of FIGS.1A-1D (SEQ ID NO:2) or which has the amino acid sequence encoded by thedeposited cDNA, as well as fragments, analogs and derivatives of suchpolypeptide.

[0042] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide of FIGS. 1A-1D (SEQ ID NO:2) or that encoded by thedeposited cDNA, means a polypeptide which either retains substantiallythe same biological function or activity as such polypeptide, i.e.functions as a G-protein coupled receptor, or retains the ability tobind the ligand or the receptor even though the polypeptide does notfunction as a G-protein coupled receptor, for example, a soluble form ofthe receptor. An analog includes a proprotein which can be activated bycleavage of the proprotein portion to produce an active maturepolypeptide.

[0043] The polypeptides of the present invention may be recombinantpolypeptides, a natural polypeptides or synthetic polypeptides,preferably recombinant polypeptides.

[0044] The fragment, derivative or analog of the polypeptide of FIGS.1A-1D (SEQ ID NO:2) or that encoded by the deposited cDNA may be (i) onein which one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide which is employed for purificationof the mature polypeptide or (v) one in which a fragment of thepolypeptide is soluble, i.e. not membrane bound, yet still binds ligandsto the membrane bound receptor. Such fragments, derivatives and analogsare deemed to be within the scope of those skilled in the art from theteachings herein.

[0045] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0046] The polypeptides of the present invention include the polypeptideof SEQ ID NO:2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptide of SEQ ID NO:2 and more preferably at least90% similarity (more preferably at least 90% identity) to thepolypeptide of SEQ ID NO:2 and still more preferably at least 95%similarity (still more preferably at least 95% identity) to thepolypeptide of SEQ ID NO:2 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

[0047] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0048] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0049] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0050] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region “leader and trailer” as well as intervening sequences(introns) between individual coding segments (exons).

[0051] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0052] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the G-protein coupled receptor genes. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0053] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0054] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0055] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0056] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0057] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0058] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowesmelanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0059] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0060] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0061] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

[0062] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0063] Fragments of the polypeptides of the present invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis, therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments ofthe polynucleotides of the present invention may be used in a similarmanner to synthesize the full-length polynucleotides of the presentinvention.

[0064] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0065] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0066] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), Â-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences. Optionally, the heterologous sequence can encodea fusion protein including an N-terminal identification peptideimparting desired characteristics, e.g., stabilization or simplifiedpurification of expressed recombinant product.

[0067] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0068] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0069] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0070] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0071] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0072] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0073] The G-protein coupled receptor polypeptide of the presentinvention can be recovered and purified from recombinant cell culturesby methods including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

[0074] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

[0075] Fragments of the full length G-protein coupled receptor genes maybe employed as a hybridization probe for a cDNA library to isolate thefull length genes and to isolate other genes which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype have at least 20 bases, preferably 30 bases and most preferably 50bases or more. The probe may also be used to identify a cDNA clonecorresponding to a fall length transcript and a genomic clone or clonesthat contain the complete G-protein coupled receptor gene includingregulatory and promotor regions, exons, and introns. As an example of ascreen comprises isolating the coding region of the G-protein coupledreceptor gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

[0076] The G-protein coupled receptors of the present invention may beemployed in a process for screening for compounds which activate(agonists) or inhibit activation (antagonists) of the receptorpolypeptide of the present invention.

[0077] In general, such screening procedures involve providingappropriate cells which express the receptor polypeptide of the presentinvention on the surface thereof. Such cells include cells from mammals,yeast, drosophila or E. Coli. In particular, a polynucleotide encodingthe receptor of the present invention is employed to transfect cells tothereby express the G-protein coupled receptor. The expressed receptoris then contacted with a test compound to observe binding, stimulationor inhibition of a functional response.

[0078] One such screening procedure involves the use of melanophoreswhich are transfected to express the G-protein coupled receptor of thepresent invention. Such a screening technique is described in PCT WO92/01810 published Feb. 6, 1992.

[0079] Thus, for example, such assay may be employed for screening for acompound which inhibits activation of the receptor polypeptide of thepresent invention by contacting the melanophore cells which encode thereceptor with both the receptor ligand and a compound to be screened.Inhibition of the signal generated by the ligand indicates that acompound is a potential antagonist for the receptor, i.e., inhibitsactivation of the receptor.

[0080] The screen may be employed for determining a compound whichactivates the receptor by contacting such cells with compounds to bescreened and determining whether such compound generates a signal, i.e.,activates the receptor.

[0081] Other screening techniques include the use of cells which expressthe G-protein coupled receptor (for example, transfected CHO cells) in asystem which measures extracellular pH changes caused by receptoractivation, for example, as described in Science, volume 246, pages181-296 (October 1989). For example, compounds may be contacted with acell which expresses the receptor polypeptide of the present inventionand a second messenger response, e.g. signal transduction or pH changes,may be measured to determine whether the potential compound activates orinhibits the receptor.

[0082] Another such screening technique involves introducing RNAencoding the G-protein coupled receptor into Xenopus oocytes totransiently express the receptor. The receptor oocytes may then becontacted with the receptor ligand and a compound to be screened,followed by detection of inhibition or activation of a calcium signal inthe case of screening for compounds which are thought to inhibitactivation of the receptor.

[0083] Another screening technique involves expressing the G-proteincoupled receptor in which the receptor is linked to a phospholipase C orD. As representative examples of such cells, there may be mentionedendothelial cells, smooth muscle cells, embryonic kidney cells, etc. Thescreening may be accomplished as hereinabove described by detectingactivation of the receptor or inhibition of activation of the receptorfrom the phospholipase second signal.

[0084] Another method involves screening for compounds which inhibitactivation of the receptor polypeptide of the present inventionantagonists by determining inhibition of binding of labeled ligand tocells which have the receptor on the surface thereof. Such a methodinvolves transfecting a eukaryotic cell with DNA encoding the G-proteincoupled receptor such that the cell expresses the receptor on itssurface and contacting the cell with a compound in the presence of alabeled form of a known ligand. The ligand can be labeled, e.g., byradioactivity. The amount of labeled ligand bound to the receptors ismeasured, e.g., by measuring radioactivity of the receptors. If thecompound binds to the receptor as determined by a reduction of labeledligand which binds to the receptors, the binding of labeled ligand tothe receptor is inhibited.

[0085] G-protein coupled receptors are ubiquitous in the mammalian hostand are responsible for many biological functions, including manypathologies. Accordingly, it is desirous to find compounds and drugswhich stimulate the G-protein coupled receptor on the one hand and whichcan inhibit the function of a G-protein coupled receptor on the otherhand.

[0086] For example, compounds which activate the G-protein coupledreceptor may be employed for therapeutic purposes, such as the treatmentof asthma, Parkinson's disease, acute heart failure, hypotension,urinary retention, and osteoporosis.

[0087] In general, compounds which inhibit activation of the G-proteincoupled receptor may be employed for a variety of therapeutic purposes,for example, for the treatment of hypertension, angina pectoris,myocardial infarction, ulcers, asthma, allergies, benign prostatichypertrophy and psychotic and neurological disorders, includingschizophrenia, manic excitement, depression, delirium, dementia orsevere mental retardation, dyskinesias, such as Huntington's disease orGilles dila Tourett's syndrome, among others. Compounds which inhibitG-protein coupled receptors have also been useful in reversingendogenous anorexia and in the control of bulimia.

[0088] An antibody may antagonize a G-protein coupled receptor of thepresent invention, or in some cases an oligopeptide, which bind to theG-protein coupled receptor but does not elicit a second messengerresponse such that the activity of the G-protein coupled receptors isprevented. Antibodies include anti-idiotypic antibodies which recognizeunique determinants generally associated with the antigen-binding siteof an antibody. Potential antagonist compounds also include proteinswhich are closely related to the ligand of the G-protein coupledreceptors, i.e. a fragment of the ligand, which have lost biologicalfunction and when binding to the G-protein coupled receptor, elicit noresponse.

[0089] An antisense construct prepared through the use of antisensetechnology, may be used to control gene expression through triple-helixformation or antisense DNA or RNA, both of which methods are based onbinding of a polynucleotide to DNA or RNA. For example, the 5′ codingportion of the polynucleotide sequence, which encodes for the maturepolypeptides of the present invention, is used to design an antisenseRNA oligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervanet al., Science, 251: 1360 (1991)), thereby preventing transcription andthe production of G-protein coupled receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofmRNA molecules into G-protein coupled receptor (antisense—Okano, J.Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of G-protein coupled receptor.

[0090] A small molecule which binds to the G-protein coupled receptor,making it inaccessible to ligands such that normal biological activityis prevented, for example small peptides or peptide-like molecules, mayalso be used to inhibit activation of the receptor polypeptide of thepresent invention.

[0091] A soluble form of the G-protein coupled receptor, e.g. a fragmentof the receptors, may be used to inhibit activation of the receptor bybinding to the ligand to a polypeptide of the present invention andpreventing the ligand from interacting with membrane bound G-proteincoupled receptors.

[0092] This invention additionally provides a method of treating anabnormal condition related to an excess of G-protein coupled receptoractivity which comprises administering to a subject the inhibitorcompounds as hereinabove described along with a pharmaceuticallyacceptable carrier in an amount effective to inhibit activation byblocking binding of ligands to the G-protein coupled receptors, or byinhibiting a second signal, and thereby alleviating the abnormalconditions.

[0093] The invention also provides a method of treating abnormalconditions related to an under-expression of G-protein coupled receptoractivity which comprises administering to a subject a therapeuticallyeffective amount of a compound which activates the receptor polypeptideof the present invention as described above in combination with apharmaceutically acceptable carrier, to thereby alleviate the abnormalconditions.

[0094] The soluble form of the G-protein coupled receptor, and compoundswhich activate or inhibit such receptor, may be employed in combinationwith a suitable pharmaceutical carrier. Such compositions comprise atherapeutically effective amount of the polypeptide or compound, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

[0095] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the pharmaceutical compositions may be employed in conjunctionwith other therapeutic compounds.

[0096] The pharmaceutical compositions may be administered in aconvenient manner such as by the topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, the pharmaceutical compositions will be administered in anamount of at least about 10 g/kg body weight and in most cases they willbe administered in an amount not in excess of about 8 mg/Kg body weightper day. In most cases, the dosage is from about 10 g/kg to about 1mg/kg body weight daily, taking into account the routes ofadministration, symptoms, etc.

[0097] The G-protein coupled receptor polypeptides, and compounds whichactivate or inhibit which are also compounds may be employed inaccordance with the present invention by expression of such polypeptidesin vivo, which is often referred to as “gene therapy.”

[0098] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

[0099] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

[0100] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0101] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990(1989), or any other promoter (e.g., cellular promoters such aseukaryotic cellular promoters including, but not limited to, thehistone, pol III, and β-actin promoters). Other viral promoters whichmay be employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

[0102] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe genes encoding the polypeptides.

[0103] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, psi-2 (ψ-2), psi-AM (ψ-AM), PA12, T19-14X, VT-19-17-H2, psiCRE(ψCRE), psiCRIP (ψCRIP), GP+E-86, GP+envAm12, and DAN cell lines asdescribed in Miller, Human Gene Therapy, Vol. 1, pg. 5-14 (1990), whichis incorporated herein by reference in its entirety. The vector maytransduce the packaging cells through any means known in the art. Suchmeans include, but are not limited to, electroporation, the use ofliposomes, and CaPO precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

[0104] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0105] The present invention also provides a method for determiningwhether a ligand not known to be capable of binding to a G-proteincoupled receptor of the present invention can bind to such receptorwhich comprises contacting a mammalian cell which expresses a G-proteincoupled receptor with the ligand under conditions permitting binding ofligands to the G-protein coupled receptor, detecting the presence of aligand which binds to the receptor and thereby determining whether theligand binds to the G-protein coupled receptor.

[0106] This invention further provides a method of screening drugs toidentify drugs which specifically interact with, and bind to, the humanG-protein coupled receptors on the surface of a cell which comprisescontacting a mammalian cell comprising an isolated DNA molecule encodingthe G-protein coupled receptor with a plurality of drugs, determiningthose drugs which bind to the mammalian cell, and thereby identifyingdrugs which specifically interact with and bind to a human G-proteincoupled receptor of the present invention. Such drugs may then be usedtherapeutically to either activate or inhibit activation of thereceptors of the present invention.

[0107] This invention also provides a method of detecting expression ofthe G-protein coupled receptor on the surface of a cell by detecting thepresence of mRNA coding for a G-protein coupled receptor which comprisesobtaining total mRNA from the cell and contacting the mRNA so obtainedwith a nucleic acid probe of the present invention capable ofspecifically hybridizing with a sequence included within the sequence ofa nucleic acid molecule encoding a human G-protein coupled receptorunder hybridizing conditions, detecting the presence of MRNA hybridizedto the probe, and thereby detecting the expression of the G-proteincoupled receptor by the cell.

[0108] This invention is also related to the use of the G-proteincoupled receptor genes as part of a diagnostic assay for detectingdiseases or susceptibility to diseases related to the presence ofmutations in the nucleic acid sequences with encode the receptorpolypeptides of the present invention. Such diseases, by way of example,are related to cell transformation, such as tumors and cancers.

[0109] Individuals carrying mutations in the human G-protein coupledreceptor gene may be detected at the DNA level by a variety oftechniques. Nucleic acids for diagnosis may be obtained from a patient'scells, such as from blood, urine, saliva, tissue biopsy and autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR (Saiki et al, Nature, 324:163-166(1986)) prior to analysis. RNA or cDNA may also be used for the samepurpose. As an example, PCR primers complementary to the nucleic acidencoding the G-protein coupled receptor proteins can be used to identifyand analyze G-protein coupled receptor mutations. For example, deletionsand insertions can be detected by a change in size of the amplifiedproduct in comparison to the normal genotype. Point mutations can beidentified by hybridizing amplified DNA to radiolabeled G-proteincoupled receptor RNA or alternatively, radiolabeled G-protein coupledreceptor antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

[0110] Sequence differences between the reference gene and gene havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

[0111] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

[0112] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0113] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0114] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0115] The present invention also relates to a diagnostic assay fordetecting altered levels of soluble forms of the receptor polypeptidesof the present invention in various tissues. Assays used to detectlevels of the soluble receptor polypeptides in a sample derived from ahost are well known to those of skill in the art and includeradioimmunoassays, competitive-binding assays, Western blot analysis andpreferably as ELISA assay.

[0116] An ELISA assay initially comprises preparing an antibody specificto antigens of the receptor polypeptide, preferably a monoclonalantibody. In addition a reporter antibody is prepared against themonoclonal antibody. To the reporter antibody is attached a detectablereagent such as radioactivity, fluorescence or in this example ahorseradish peroxidase enzyme. A sample is now removed from a host andincubated on a solid support, e.g. a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein such as bovineserum albumin. Next, the monoclonal antibody is incubated in the dishduring which time the monoclonal antibodies attach to any receptorpolypeptides of the present invention attached to the polystyrene dish.All unbound monoclonal antibody is washed out with buffer. The reporterantibody linked to horseradish peroxidase is now placed in the dishresulting in binding of the reporter antibody to any monoclonal antibodybound to receptor proteins. Unattached reporter antibody is then washedout. Peroxidase substrates are then added to the dish and the amount ofcolor developed in a given time period is a measurement of the amount ofreceptor proteins present in a given volume of patient sample whencompared against a standard curve.

[0117] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with gene associated with disease.

[0118] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0119] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0120] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York (1988).

[0121] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0122] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0123] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0124] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0125] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0126] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0127] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0128] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0129] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0130] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0131] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0132] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980).

[0133] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0134] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units to T4 DNAligase (“ligase” ) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0135] Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

EXAMPLE 1 Expression of Recombinant HGBER32 in COS 7 Cells

[0136] The expression of plasmid, HGBER32 HA is derived from a vectorpcDNA3 (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E. coli replication origin, 4) CMVpromoter followed by a polylinker region, a SV40 intron andpolyadenylation site. A DNA fragment encoding the entire HGBER32precursor and a HA tag fused in frame to its 3′ end was cloned into thepolylinker region of the vector, therefore, the recombinant proteinexpression is directed by the CMV promoter. The HA tag corresponds to anepitope derived from the influenza hemagglutinin protein as previouslydescribed (Wilson et al., Cell, 37:767, 1984). The infusion of HA tag tothe target protein allows easy detection of the recombinant protein withan antibody that recognizes the HA epitope.

[0137] The plasmid construction strategy is described as follows:

[0138] The DNA sequence encoding HGBER32, ATCC No. 97187, wasconstructed by PCR on a genomic lambda clone using two primers: the 5′primer; 5 ACCAGGATCCGCTGCCTTGATGGATTAT′ (SEQ ID NO:4) contains a BAMHIsite followed by 9 nucleotides of HGBER32 coding sequence starting fromthe initiation codon; the 3′ primer (SEQ ID NO:5) 5CTGCTTCTAGAATGCCATTCAAGAAAATGTT contains complementary sequences to XbaIsite, translation stop codon, 10 nucleotides of the HGBER32 codingsequence (not including the stop codon). Therefore, the PCR productcontains a BAMHI site, HGBER32 coding sequence followed by a translationtermination stop codon and an XbaI site. The PCR amplified DNA fragmentand the vector, pcDNAI/Amp, were digested with BAMHI and XbaIrestriction enzyme and ligated. The ligation mixture was transformedinto E. coli strain SURE (available from Stratagene Cloning Systems,11099 North Torrey Pines Road, La Jolla, Calif. 92037) the transformedculture was plated on ampicillin media plates and resistant colonieswere selected. Plasmid DNA was isolated from transformants and examinedby restriction analysis for the presence of the correct fragment. Forexpression of the recombinant HGBER32, COS 7 cells were transfected withthe expression vector by DEAE-DEXTRAN method (Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring LaboratoryPress, 1989). The expression of the HGBER32 HA protein was detected byradiolabelling and immunoprecipitation method (Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,1988). Cells were labelled for 8 hours with ³⁵S-cysteine two days posttransfection. Culture media were then collected and cells were lysedwith detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40,0.5% DOC, 50mM Tris, pH 7.5) (Wilson et al., Id., 37:767, 1984). Bothcell lysate and culture media were precipitated with a HA specificmonoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGEgels.

EXAMPLE 2 Cloning and Expression of HGBER32 Using the BaculovirusExpression System

[0139] The DNA sequence encoding the full length HGBER32 protein, ATCCNo. 97187, was amplified using PCR oligonucleotide primers correspondingto the 5′ and 3′ sequences of the gene:

[0140] The 5′ primer has the sequenceGTGACCGGATCCCGCTGCCTTGCCGCCATCATGGATTATACACTTGACCTCAGTG (SEQ ID NO:6)and contains a BAMHI restriction enzyme site (in bold) followed by 18nucleotides resembling an efficient signal for the initiation oftranslation in eukaryotic cells (Kozak, J Mol. Biol., 196:947-950,1987), and just behind the first 25 nucleotides of the HGBER32 gene (theinitiation codon for translation “ATG” is underlined).

[0141] The 3′ primer has the sequence TTAATCTAGAGTCTTCATTGATCCTCCCAAATG(SEQ ID NO:7) and contains the cleavage site for the restrictionendonuclease XbaI and 4 nucleotides complementary to the 3′non-translated sequence of the HGBER32 gene. The amplified sequenceswere isolated from a 1% agarose gel using a commercially available kit(“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment was thendigested with the endonucleases BAMHI and XbaI and then purified asdescribed in Example 1. This fragment is designated F2.

[0142] The vector pA2 (modification of PUL941 vector, discussed below)is used for the expression of the HGBER32 protein using the baculovirusexpression system (for review see: Summers and Smith, A Manual ofMethods for Baculovirus Vectors and Insect Cell Culture Procedures,Texas Agricultural Experimental Station Bulletin No. 1555, 1987). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BAMHI and XbaI.The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusesthe beta-galactosidase gene from E. coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pA2 such as pRG1, pAc373,pVL941 and pAcIM1 (Luckow and Summers, Virology, 170:31-39).

[0143] The plasmid was digested with the restriction enzymes BAMHI andXbaI and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The DNA was then isolated from a 1% agarosegel as described in Example 1. This vector DNA is designated V2.

[0144] Fragment F2 and the dephosphorylated plasmid V2 were ligated withT4 DNA ligase. E.coli HB101 cells were then transformed and bacteriaidentified that contained the plasmid (pBac-HGBER32) with the HGBER32gene. The sequence of the cloned fragment was confirmed by DNAsequencing.

[0145] 5 μg of the plasmid pBac-HGPCR were co-transfected with 1.0 μg ofa commercially available linearized baculovirus (“BaculoGold baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al., Proc. Natl Acad. Sci. USA, 84:7413-7417 (1987)).

[0146] 1 μg of BaculoGold virus DNA and 5 μg of the plasmid pBac-HGBER32were mixed in a sterile well of a microtiter plate containing 50 μl ofserum free Grace's medium (Life Technologies Inc., Gaithersburg, Md).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium were added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture was added drop wise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace' medium withoutserum. The plate was rocked back and forth to mix the newly addedsolution. The plate was then incubated for 5 hours at 27° C. After 5hours the transfection solution was removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum was added.The plate was put back into an incubator and cultivation continued at27° C. for four days.

[0147] After four days the supernatant was collected and a plaque assaywas performed in a manner similar to that described by Summers andSmith, supra. As a modification, an agarose gel with “Blue Gal” (LifeTechnologies Inc., Gaithersburg) was used which allows an easy isolationof blue stained plaques. (A detailed description of a “plaque assay” canalso be found in the user's guide for insect cell culture andbaculovirology distributed by Life Technologies Inc., Gaithersburg, page9-10).

[0148] Four days after the serial dilution, the viruses were added tothe cells and blue stained plaques were picked with the tip of anEppendorf pipette. The agar containing the recombinant viruses was thenresuspended in an Eppendorf tube containing 200 μl of Grace's medium.The agar was removed by a brief centrifugation and the supernatantcontaining the recombinant baculoviruses was used to infect Sf9 cellsseeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then stored at 4° C.

[0149] Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-HGBER32 at a multiplicity of infection (MOI) of 2. Sixhours later the medium was removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S cysteine (Amersham)were added. The cells were further incubated for 16 hours before theywere harvested by centrifugation and the labelled proteins werevisualized by SDS-PAGE and autoradiography.

EXAMPLE 3 Expression Pattern of HGBER32 in Human Tissue

[0150] Northern blot analysis is carried out to examine the levels ofexpression of HGBER32 in human tissues. Total cellular RNA samples areisolated with RNAzol B system (Biotecx Laboratories, Inc. 6023 SouthLoop East, Houston, Tex. 77033). About 10 g of total RNA isolated fromeach human tissue specified is separated on 1% agarose gel and blottedonto a nylon filter. (Sambrook et al., supra. The labeling reaction isdone according to the Stratagene Prime-It kit with 50 ng DNA fragment.The labeled DNA is purified with a Select-G-50 column. (5 Prime—3 Prime,Inc. 5603 Arapahoe Road, Boulder, Colo. 80303). The filter is thenhybridized with radioactive labeled full length HGBER32 gene at1,000,000 cpm/ml in 0.5 M NaPO, pH 7.4 and 7% SDS overnight at 65 C.After being washed twice at room temperature and twice at 60 C with0.5×SSC, 0.1% SDS, the filter is then exposed at −70 C overnight with anintensifying screen. The messenger RNA for HGBER32 is abundant in humancerebellum tissue.

EXAMPLE 4 Expression via Gene Therapy

[0151] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0152] pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked bythe long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0153] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer containing an EcoRI site and the3′ primer having contains a HindIII site. Equal quantities of theMoloney murine sarcoma virus linear backbone and the EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

[0154] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0155] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his.

[0156] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

[0157] Numerous modifications and variations of the present inventionwere possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1 7 1586 base pairs nucleic acid single linear cDNA CDS 431..1495 1CCTCTTTGGG GTCCAAGTGA ATCCTTCTGC CTCAGCCTCC TGAGTAGCTA GGATTACAGG 60CATGCACCCG CCATGCCCGG CTAATTTTTG TAATTTTTAG TAGAGACGGG GTTTCCCCAT 120GTTGCCAAGG CTGGTCTTGA ACCCCTGACC TCAGGTGATC TGCCTCACCT TGGCCTCCCA 180AAGTGCTAGG ATTACAGGCA TGAGCCACAG CTCCCGGTCT ATCATTTAAC CTTAATTACA 240TCTTTAAAGG CCCAAATAGT CTCACCCACT CCAAATAGTC ACACCCACAC CGGAGGTTGA 300GCACTTCAAC ACATGAATTT GGGGAGGACA CAGTTCAGTC CATAACATCC CCCTAATTTT 360TAAAAAATAA AAATGTTTTT AAGGAGTGAA TGTCTTTTAT GTGTCTCTGT GACCAGGTCC 420CGCTGCCTTG ATG GAT TAT ACA CTT GAC CTC AGT GTG ACA ACA GTG ACC 469 MetAsp Tyr Thr Leu Asp Leu Ser Val Thr Thr Val Thr 1 5 10 GAC TAC TAC TACCCT GAT ATC TTC TCA AGC CCC TGT GAT GCG GAA CTT 517 Asp Tyr Tyr Tyr ProAsp Ile Phe Ser Ser Pro Cys Asp Ala Glu Leu 15 20 25 ATT CAG ACA AAT GGCAAG TTG CTC CTT GCT GTC TTT TAT TGC CTC CTG 565 Ile Gln Thr Asn Gly LysLeu Leu Leu Ala Val Phe Tyr Cys Leu Leu 30 35 40 45 TTT GTA TTC AGT CTTCTG GGA AAC AGC CTG GTC ATC CTG GTC CTT GTG 613 Phe Val Phe Ser Leu LeuGly Asn Ser Leu Val Ile Leu Val Leu Val 50 55 60 GTC TGC AAG AAG CTG AGGAGC ATC ACA GAT GTA TAC CTC TTG AAC CTG 661 Val Cys Lys Lys Leu Arg SerIle Thr Asp Val Tyr Leu Leu Asn Leu 65 70 75 GCC CTG TCT GAC CTG CTT TTTGTC TTC TCC TTC CCC TTT CAG ACC TAC 709 Ala Leu Ser Asp Leu Leu Phe ValPhe Ser Phe Pro Phe Gln Thr Tyr 80 85 90 TAT CTG CTG GAC CAG TGG GTG TTTGGG ACT GTA ATG TGC AAA GTG GTG 757 Tyr Leu Leu Asp Gln Trp Val Phe GlyThr Val Met Cys Lys Val Val 95 100 105 TCT GGC TTT TAT TAC ATT GGC TTCTAC AGC AGC ATG TTT TTC ATC ACC 805 Ser Gly Phe Tyr Tyr Ile Gly Phe TyrSer Ser Met Phe Phe Ile Thr 110 115 120 125 CTC ATG AGT GTG GAC AGG TACCTG GCT GTT GTC CAT GCC GTG TAT GCC 853 Leu Met Ser Val Asp Arg Tyr LeuAla Val Val His Ala Val Tyr Ala 130 135 140 CTA AAG GTG AGG ACG ATC AGGATG GGC ACA ACG CTG TGC CTG GCA GTA 901 Leu Lys Val Arg Thr Ile Arg MetGly Thr Thr Leu Cys Leu Ala Val 145 150 155 TGG CTA ACC GCC ATT ATG GCTACC ATC CCA TTG CTA GTG TTT TAC CAA 949 Trp Leu Thr Ala Ile Met Ala ThrIle Pro Leu Leu Val Phe Tyr Gln 160 165 170 GTG GCC TCT GAA GAT GGT GTTCTA CAG TGT TAT TCA TTT TAC AAT CAA 997 Val Ala Ser Glu Asp Gly Val LeuGln Cys Tyr Ser Phe Tyr Asn Gln 175 180 185 CAG ACT TTG AAG TGG AAG ATCTTC ACC AAC TTC AAA ATG AAC ATT TTA 1045 Gln Thr Leu Lys Trp Lys Ile PheThr Asn Phe Lys Met Asn Ile Leu 190 195 200 205 GGC TTG TTG ATC CCA TTCACC ATC TTT ATG TTC TGC TAC ATT AAA ATC 1093 Gly Leu Leu Ile Pro Phe ThrIle Phe Met Phe Cys Tyr Ile Lys Ile 210 215 220 CTG CAC CAG CTG AAG AGGTGT CAA AAC CAC AAC AAG ACC AAG GCC ATC 1141 Leu His Gln Leu Lys Arg CysGln Asn His Asn Lys Thr Lys Ala Ile 225 230 235 AGG TTG GTG CTC ATT GTGGTC ATT GCA TCT TTA CTT TTC TGG GTC CCA 1189 Arg Leu Val Leu Ile Val ValIle Ala Ser Leu Leu Phe Trp Val Pro 240 245 250 TTC AAC GTG GTT CTT TTCCTC ACT TCC TTG CAC AGT ATG CAC ATC TTG 1237 Phe Asn Val Val Leu Phe LeuThr Ser Leu His Ser Met His Ile Leu 255 260 265 GAT GGA TGT AGC ATA AGCCAA CAG CTG ACT TAT GCC ACC CAT GTC ACA 1285 Asp Gly Cys Ser Ile Ser GlnGln Leu Thr Tyr Ala Thr His Val Thr 270 275 280 285 GAA ATC ATT TCC TTTACT CAC TGC TGT GTG AAC CCT GTT ATC TAT GCT 1333 Glu Ile Ile Ser Phe ThrHis Cys Cys Val Asn Pro Val Ile Tyr Ala 290 295 300 TTT GTT GGG GAG AAGTTC AAG AAA CAC CTC TCA GAA ATA TTT CAG AAA 1381 Phe Val Gly Glu Lys PheLys Lys His Leu Ser Glu Ile Phe Gln Lys 305 310 315 AGT TGC AGC CAA ATCTTC AAC TAC CTA GGA AGA CAA ATG CCT AGG GAG 1429 Ser Cys Ser Gln Ile PheAsn Tyr Leu Gly Arg Gln Met Pro Arg Glu 320 325 330 AGC TGT GAA AAG TCATCA TCC TGC CAG CAG CAC TCC TCC CGT TCC TCC 1477 Ser Cys Glu Lys Ser SerSer Cys Gln Gln His Ser Ser Arg Ser Ser 335 340 345 AGC GTA GAC TAC ATTTTG TAGGATCAAT GAAGACTAAA TATTAAAAAC 1525 Ser Val Asp Tyr Ile Leu 350355 ATTTNCTTGA ATGGNATGCT AGTAGCAGNG GAGCAAAGGT GTGGGTGTGA AAGGTTTC 1585A 1586 355 amino acids amino acid linear protein 2 Met Asp Tyr Thr LeuAsp Leu Ser Val Thr Thr Val Thr Asp Tyr Tyr 1 5 10 15 Tyr Pro Asp IlePhe Ser Ser Pro Cys Asp Ala Glu Leu Ile Gln Thr 20 25 30 Asn Gly Lys LeuLeu Leu Ala Val Phe Tyr Cys Leu Leu Phe Val Phe 35 40 45 Ser Leu Leu GlyAsn Ser Leu Val Ile Leu Val Leu Val Val Cys Lys 50 55 60 Lys Leu Arg SerIle Thr Asp Val Tyr Leu Leu Asn Leu Ala Leu Ser 65 70 75 80 Asp Leu LeuPhe Val Phe Ser Phe Pro Phe Gln Thr Tyr Tyr Leu Leu 85 90 95 Asp Gln TrpVal Phe Gly Thr Val Met Cys Lys Val Val Ser Gly Phe 100 105 110 Tyr TyrIle Gly Phe Tyr Ser Ser Met Phe Phe Ile Thr Leu Met Ser 115 120 125 ValAsp Arg Tyr Leu Ala Val Val His Ala Val Tyr Ala Leu Lys Val 130 135 140Arg Thr Ile Arg Met Gly Thr Thr Leu Cys Leu Ala Val Trp Leu Thr 145 150155 160 Ala Ile Met Ala Thr Ile Pro Leu Leu Val Phe Tyr Gln Val Ala Ser165 170 175 Glu Asp Gly Val Leu Gln Cys Tyr Ser Phe Tyr Asn Gln Gln ThrLeu 180 185 190 Lys Trp Lys Ile Phe Thr Asn Phe Lys Met Asn Ile Leu GlyLeu Leu 195 200 205 Ile Pro Phe Thr Ile Phe Met Phe Cys Tyr Ile Lys IleLeu His Gln 210 215 220 Leu Lys Arg Cys Gln Asn His Asn Lys Thr Lys AlaIle Arg Leu Val 225 230 235 240 Leu Ile Val Val Ile Ala Ser Leu Leu PheTrp Val Pro Phe Asn Val 245 250 255 Val Leu Phe Leu Thr Ser Leu His SerMet His Ile Leu Asp Gly Cys 260 265 270 Ser Ile Ser Gln Gln Leu Thr TyrAla Thr His Val Thr Glu Ile Ile 275 280 285 Ser Phe Thr His Cys Cys ValAsn Pro Val Ile Tyr Ala Phe Val Gly 290 295 300 Glu Lys Phe Lys Lys HisLeu Ser Glu Ile Phe Gln Lys Ser Cys Ser 305 310 315 320 Gln Ile Phe AsnTyr Leu Gly Arg Gln Met Pro Arg Glu Ser Cys Glu 325 330 335 Lys Ser SerSer Cys Gln Gln His Ser Ser Arg Ser Ser Ser Val Asp 340 345 350 Tyr IleLeu 355 347 amino acids amino acid <Unknown> linear protein 3 Asn GluSer Gly Glu Glu Val Thr Thr Phe Phe Asp Tyr Asp Tyr Gly 1 5 10 15 AlaPro Cys His Lys Phe Asp Val Lys Gln Ile Gly Ala Gln Leu Leu 20 25 30 ProPro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met 35 40 45 LeuVal Val Leu Ile Leu Ile Asn Cys Lys Lys Leu Lys Cys Leu Thr 50 55 60 AspIle Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Leu Phe Leu Ile 65 70 75 80Thr Leu Pro Leu Trp Ala His Ser Ala Ala Asn Glu Trp Val Phe Gly 85 90 95Asn Ala Met Cys Lys Leu Phe Thr Gly Leu Tyr His Ile Gly Tyr Phe 100 105110 Gly Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala 115120 125 Ile Val His Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe Gly130 135 140 Val Val Thr Ser Val Ile Thr Trp Leu Val Ala Val Phe Ala SerVal 145 150 155 160 Pro Gly Ile Ile Phe Thr Lys Cys Gln Lys Glu Asp SerVal Tyr Val 165 170 175 Cys Gly Pro Tyr Phe Pro Arg Gly Trp Asn Asn PheHis Thr Ile Met 180 185 190 Arg Asn Ile Leu Gly Leu Val Leu Pro Leu LeuIle Met Val Ile Cys 195 200 205 Tyr Ser Gly Ile Leu Lys Thr Leu Leu ArgCys Arg Asn Glu Lys Lys 210 215 220 Arg His Arg Ala Val Arg Val Ile PheThr Ile Met Ile Val Tyr Phe 225 230 235 240 Leu Phe Trp Thr Pro Tyr AsnIle Val Ile Leu Leu Asn Thr Phe Gln 245 250 255 Glu Phe Phe Gly Leu SerAsn Cys Glu Ser Thr Ser Gln Leu Asp Gln 260 265 270 Ala Thr Gln Val ThrGlu Thr Leu Gly Met Thr His Cys Cys Ile Asn 275 280 285 Pro Ile Ile TyrAla Phe Val Gly Glu Lys Phe Arg Arg Tyr Leu Ser 290 295 300 Val Phe PheArg Lys His Ile Thr Lys Arg Phe Cys Lys Gln Cys Pro 305 310 315 320 ValPhe Tyr Arg Glu Thr Val Asp Gly Val Thr Ser Thr Asn Thr Pro 325 330 335Ser Thr Gly Glu Gln Glu Val Ser Ala Gly Leu 340 345 28 base pairsnucleic acid single linear other nucleic acid /desc = “PRIMER” 4ACCAGGATCC GCTGCCTTGA TGGATTAT 28 31 base pairs nucleic acid singlelinear other nucleic acid /desc = “PRIMER” 5 CTGCTTCTAG AATGCCATTCAAGAAAATGT T 31 55 base pairs nucleic acid single linear other nucleicacid /desc = “PRIMER” 6 GTGACCGGAT CCCGCTGCCT TGCCGCCATC ATGGATTATACACTTGACCT CAGTG 55 33 base pairs nucleic acid single linear othernucleic acid /desc = “PRIMER” 7 TTAATCTAGA GTCTTCATTG ATCCTCCCAA ATG 33

What is claimed is:
 1. An isolated polynucleotide comprising a memberselected from the group consisting of: (a) a polynucleotide encoding thepolypeptide as set forth in FIGS. 1A-1D. (b) a polynucleotide encodingthe polypeptide expressed by the DNA contained in ATCC Deposit No.97187; (c) a polynucleotide capable of hybridizing to and which is atleast 70% identical to the polynucleotide of (a) or (b); and (d) apolynucleotide fragment of the polynucleotide of (a), (b) or (c).
 2. Thepolynucleotide of claim 1 encoding the polypeptide of FIGS. 1A-1D. 3.The polynucleotide of claim 1 wherein said polynucleotide encodes amature polypeptide encoded by the DNA contained in ATCC Deposit No.97187.
 4. A vector containing the polynucleotide of claim
 1. 5. A hostcell genetically engineered with the vector of claim
 4. 6. A process forproducing a polypeptide comprising: expressing from the host cell ofclaim 5 the polypeptide encoded by said polynucleotide.
 7. A process forproducing cells capable of expressing a polypeptide comprisinggenetically engineering cells with the vector of claim
 4. 8. Apolypeptide selected from the group consisting of (i) a polypeptidehaving the deduced amino acid sequence of FIGS. 1A-1D and fragments,analogs and derivatives thereof; and (ii) a polypeptide encoded by thecDNA of ATCC Deposit No. 97187 and fragments, analogs and derivatives ofsaid polypeptide.
 9. The polypeptide of claim 8 wherein the polypeptidehas the deduced amino acid sequence of FIGS. 1A-1D.
 10. An antibodyagainst the polypeptide of claim
 8. 11. A compound which activates thepolypeptide of claim
 8. 12. A compound which inhibits activation of thepolypeptide of claim
 8. 13. A method for the treatment of a patienthaving need to activate a receptor comprising: administering to thepatient a therapeutically effective amount of the compound of claim 11.14. A method for the treatment of a patient having need to inhibit areceptor comprising: administering to the patient a therapeuticallyeffective amount of the compound of claim
 12. 15. The method of claim 13wherein said compound is a polypeptide and a therapeutically effectiveamount of the compound is administered by providing to the patient DNAencoding said agonist and expressing said agonist in vivo.
 16. Themethod of claim 14 wherein said compound is a polypeptide and atherapeutically effective amount of the compound is administered byproviding to the patient DNA encoding said antagonist and expressingsaid antagonist in vivo.
 17. A method for identifying a compound whichbind to and activate the polypeptide of claim 8 comprising: contacting acompound with cells expressing on the surface thereof the polypeptide ofclaim 8, said polypeptide being associated with a second componentcapable of providing a detectable signal in response to the binding of acompound to said polypeptide said contacting being under conditionssufficient to permit binding of compounds to the polypeptide; andidentifying a compound capable of polypeptide binding by detecting thesignal produced by said second component.
 18. A method for identifyingcompounds which bind to and inhibit activation of the polypeptide ofclaim 8 comprising: contacting an analytically detectable ligand knownto bind to the receptor polypeptide and a compound with host cellsexpressing on the surface thereof the polypeptide of claim 8, saidpolypeptide being associated with a second component capable ofproviding a detectable signal in response to the binding of a compoundto said polypeptide under conditions to permit binding to thepolypeptide; and determining whether the ligand binds to the polypeptideby detecting the absence of a signal generated from the interaction ofthe ligand with the polypeptide.
 19. A process for diagnosing in apatient a disease or a susceptibility to a disease related to anunder-expression of the polypeptide of claim 8 comprising: determining amutation in the nucleic acid sequence encoding said polypeptide, or theamount of the polypeptide in a sample derived from a patient.